1. Field Of The Invention
This invention relates to the creation, optimization and use of new enzyme catalysts for deprotection in organic synthesis. More specifically, the invention relates to enzymes optimized to remove ester-linked para-nitrobenzyl (pNB) protecting groups from carboxyl functional groups on .beta.-lactam antibiotics and other compounds. This invention also relates to methods by which such enzymes can be altered and optimized for specific substrates and reaction conditions.
2. Description Of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and identified in the appended bibliography.
Efficient protection and deprotection of functional groups is critical to successful organic synthesis of polyfunctional molecules. Synthetic schemes often require that a given functional group be protected or deprotected selectively, under the mildest conditions and in the presence of functional groups of similar reactivity or other structures that are sensitive to acids, bases, oxidation and reduction. These situations represent severe problems for the synthesis of complex, polyfunctional molecules which cannot, or only with great difficulty, be solved using classical chemical tools.
The array of protecting group techniques can be substantially enriched by the application of enzymes. Enzymes can discriminate stereoisomers as well as offer the opportunity to carry out highly chemo- and regioselective transformations. The highly selective nature of enzymes may be exploited advantageously in the manipulation of protecting groups and in the synthesis of chiral compounds such as drugs and natural products. Furthermore, enzymes function under mild conditions, often operating at or near room temperature and at neutral, weakly acidic or weakly basic pH values. In many cases they combine a high selectivity for the reactions they catalyze and the structures they recognize with a broad substrate tolerance. Therefore, the application of enzymes can be viable alternatives to classical chemical protection/deprotection methods for the introduction and/or removal of suitable protecting groups (1). The introduction of new enzymes with reactivities and substrate tolerances differing from existing enzymes is highly desirable.
Carboxy groups are often protectel by conversion to the benzyl or para-nitrobenzyl (pNB) esters (2). Benzyl esters are resistant to treatment with reagents such as trifluoroacetic acid, triethylamine, diisopropylethylamine, but are readily removed by hydrogenolysis (over a Pd catalyst). Hydrogenolysis is not appropriate, however, for compounds containing double bonds, azides, imines, or activated aldehydes, or other functional groups that will be reduced. Benzyl esters can also be cleaved using a zinc catalyst under anhydrous conditions, but the extent of hydrolysis is variable and dependent on the conditions (e.g., time and temperature) of the reaction. The reaction must be carried out under anhydrous conditions, in an organic solvent. Both the organic solvent and catalyst can give rise to toxicity or disposal problems for large-scale reactions.
Modification by substitution in the aromatic ring can alter the sensitivity of the benzyl group towards deprotection by acidic reagents. PNB esters display increased resistance to acid hydrolysis.
During total synthesis or chemical modification of an antibiotic, several sites on the antibiotic could be adversely affected by the reagents used to carry out any given reaction step. Para-nitrobenzyl alcohol (pNB--OH) is commonly used to protect carboxylic acid functionalities in exphalosporin-derived antibiotics (U.S. Pat. No. 3,725,359 1975!) (3). The pNB ester linkage is stable enough to withstand the various reaction conditions used in subsequent chemical steps. After chemical synthesis is completed, deprotection is required to return the cephalosporin-pNB ester to its original and active carboxylic acid form. The chemistry used to deprotect the carboxylic acid involves a catalytic form of zinc in concentrated organic solvent, and on an industrial scale this process generates large amounts of solvent and zinc-containing waste material. Cost is associated with processing of waste to make it safe for disposal. In 1975, scientists at Eli Lilly & Co. interested in pursuing alternative methods of deprotection for higher yield and lower disposal costs began a search for an esterase capable of performing this deprotection reaction (3).
The enzyme known as para-nitr)benzyl esterase (pNB esterase) was discovered in 1975 by scientists at Eli lilly & Co., who screened whole cell preparations of numerous bacterial and fung,al cultures for those possessing catalytic activity toward the hydrolysis of a p-nitrcbenzyl protected cephalosporin (3). A Bacillus subtilis culture (NRRL B8079) showed the highest catalytic activity toward two cephalosporin-derived pNB-protected substrates of all the cultures tested. Although the reaction yield was high, the enzyme activity was not sufficient to consider for industrial application.
A chromatographically pure solution of pNB esterase was isolated at Eli Lilly, and its amino acid sequence partially determined. Using this partial sequence, DNA primers were constructed and used to isolate and sequence the gene for pNB esterase. This gene was cloned into E. coli, where it was over-expressed to produce pNB esterase in large quantities (4). However, partially purified enzyme preparations of "pNB esterase" could not compete with the speed, economy, or the small reaction volumes (due to lack of solubility of substrate in purely aqueous environments) of the zinccatalyzed deprotection reaction.
The targeted reaction substrates have changed over the fifteen year period as well. Cephalosporin-derived antibiotics continued to evolve from the first generation cephalexin (one of the two original cephalosporin substrates used to search for pNB esterase), second generation cefaclor, third generation cefixime, and fourth generation loracarbef. These antibiotics have been developed to be readily absorbed (generation one), more potent (generation two), much more potent (generation three), and, finally, immensely more stable in the body (generation four) (5). They all are synthesized using the pNB ester protecting group (6). In protected form, with perhaps the exception of cefixime, all are only sparingly soluble in water.
The pNB esterase enzyme has been further characterized (6). It is a water soluble, monomeric serine esterase of 54 kD molecular weight and a pI of 4.1. The enzyme is active on a variety of ester substrates, ranging from the cephalosporin-derived compounds on which it was screened to a number of simple organic esters. Reported KM values for cephalosporin-derived substrates are in 0.5 to 2 mM range. The enzyme functions best at temperatures below 50.degree. C., and its pH optimum is between 8 and 9.
The pNB esterase still suffers from a problem common to a large number of enzyme reactions in the performance of synthetic chemistry: the desired substrates are only sparingly soluble in water, and the enzyme's catalytic ability is drastically reduced by even small quantities of water miscible non-aqueous solvents.
In view of the above problems with the existing pNB esterase, there is a continuing need to develop new enzymes which have expanded catalytic capabilities. In particular, new enzymes are needed which can be used to provide ester cleavage for a variety of substrates and settings, including polar non-aqueous solvents.